Motoneuronotrophic factors

ABSTRACT

An isolated polynucleotide encoding motoneuronotrophic factor F6.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/928,862, filed Sep. 12, 1997, now U.S. Pat. No. 6,309,877B1, which isa continuation-in-part of U.S. patent application Ser. No. 08/751,225,filed Nov. 15, 1996, now abandoned; which is a continuation in part ofU.S. provisional patent application No. 60/026,792, filed on Sep. 27,1996, now abandoned; which are hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to the human genes which encode aspecialized group of proteins which promote the growth, maintenance,survival, and functional capabilities of selected populations ofneurons.

BACKGROUND OF INVENTION

Neuronotrophic factors (NTFs) are a specialized group of proteins whichfunction to promote the survival, growth, maintenance, and functionalcapabilities of selected populations of neurons. Recent studies havedemonstrated that neuronal death occurs in the nervous systems ofvertebrates during certain periods of growth and development. However,the addition of soluble neuronal trophic factors from associated targettissues serves to mitigate this phenomenon of neuronal death. Thefollowing citations discuss neuronal trophic factors and theirdisclosures are hereby incorporated by reference: Chau, R. M. W., etal., Neuronotrophic Factor, 6 Chin. J. Neuroanatomy 129 (1990); Kuno,M., Target Dependence of Motoneuronal Survival: The Current Status, 9Neurosci. Res. 155 (1990); Bard, Y. A., Trophic Factors and NeuronalSurvival, 2 Neuron 1525 (1989); Oppenheim, R. W., The NeurotrophicTheory and Naturally Occurring Motoneuron Death, 12 TINS 252 (1989);Bard, Y. A., What, If Anything, is a Neurotrophic Factor?, 11 TINS 343(1988); and Thoenen, H., and Edgar, D., Neurotrophic Factors, 229Science 238 (1985).

In the vertebrate neuromuscular system, the survival of embryonicmotoneurons have been found to be dependent upon specific trophicsubstances derived from the associated developing skeletal muscles.Skeletal muscles have been shown, by both in vivo and in vitro studies,to produce substances which are capable of enhancing the survival anddevelopment of motoneurons by preventing the embryonic motoneurons fromdegeneration and subsequent, natural cellular death. See O'Brian, R. J.and Fischbach, G. D., Isolation of Embryonic Chick Motoneurons and TheirSurvival In Vitro, 6 J. Neurosci. 3265 (1986); Hollyday, M. andHamburger, V., Reduction of the Naturally Occurring Motor Neuron Loss byEnlargement of the Periphery, 170 J. Comp. Neurol. 311 (1976), whosedisclosures are incorporated herein by reference. Similarly, severalinvestigators have reported that chick and rat skeletal muscles possesscertain trophic factors which can prevent the natural cellular death ofembryonic motoneurons both in vivo and in vitro. See McManaman, J. L.,et al., Purification of a Skeletal Muscle Polypeptide Which StimulatesCholine Acetyltransferase Activity in Cultured Spinal Cord Neurons, 263J.Biol. Chem. 5890 (1988); Oppenheim, R. W., et al., Reduction ofNaturally Occurring Motoneuron Death In Vitro by a Target DerivedNeurotrophic Factor, 240 Science 919 (1988); and Smith, R. G., et al.,Selective Effects of Skeletal Muscle Extract Fractions on MotoneuronsDevelopment In Vivo, 6 J. Neurosci. 439 (1986), whose disclosures areincorporated herein by reference.

In addition, a polypeptide has been isolated from rat skeletal musclewhich has been found to selectively enhance the survival of embryonicchick motoneurons in vivo, as well the activity of cholineacetyltransferase in these motoneurons. This polypeptide has been namedCholine Acetyltransferase Development Factor (CDF) and its biologicalfunction has been demonstrated to be different from other trophicfactors such as Nerve Growth Factor (NGF), Ciliary Ganglion NeurotrophicFactor (CNTF), Brain-Derived Neurotrophic Factor (BDNF), and RetinalGanglion Neurotrophic Factor (RGNTF). See Levi-Montalcini, R.,“Developmental Neurobiology and the Natural History of Nerve GrowthFactor,” 5 Ann. Rev. Neurosci. 341 (1982); Varon, S., et al., GrowthFactors. In: Advances in Neurology, Vol. 47: Functional Recovery inNeurological Disease, Waxman, S. G. (ed.), Raven Press, New York, pp.493-521 (1988); Barde, Y. A., Trophic Factors and Neuronal Survival, 2Neuron 1525 (1989); Chau, R. M. W., et al., The Effect of a 30 kDProtein from Tectal Extract of Rat on Cultured Retinal Neurons, 34Science in China, Series B, 908 (1991), whose disclosures areincorporated herein by reference.

The inventor of the invention disclosed in the instant application, Dr.Raymond Ming Wah Chau, presented results which reported the isolationand characterization of two motoneuronotrophic factors from rat muscletissue having apparent molecular weights of 35 kD and 22 kD. This datawas initially disseminated in 1991 at the World Congress of Cellular andMolecular Biology held in Paris, France. See Chau, R. M. W., et al.,Muscle Neuronotrophic Factors Specific for Anterior Horn Motoneurons ofRat Spinal Cord. In: Recent Advances in Cellular and Molecular Biology,Vol. 5, Peeters Press, Leuven, Belgium, pp. 89-94 (1992), the disclosureof which is hereby incorporated by reference. This 35 kD protein hasbeen defined by Dr. Chau as motoneuronotrophic factor 1(MNTF1) and theapparent 22 kD protein as motoneuronotrophic factor 2 (MNTF2). These twotrophic factors have been demonstrated in vitro by Dr. Chau to supportthe growth and/or regeneration of both isolated anterior hornmotoneurons and spinal explants of rat lumber spinal cord.

Subsequently, in 1993, Dr. Chau reported the successful cloning of humanMNTF1, a protein having an apparent molecular weight of 55 kD, and itsassociated receptor from a human retinoblastoma cDNA library. See Chau,R. M. W., et al., Cloning of Genes for Muscle-Derived MotoneuronotrophicFactor 1 (MNTF1) and Its Receptor by Monoclonal Antibody Probes,(abstract) 19 Soc. for Neurosci. part 1, 252 (1993), the disclosure ofwhich is hereby incorporated by reference. The cloned human MNTF1 wasdemonstrated to have biological activity similar to that of the “native”MNTF1 protein in that it supported the in vitro growth of rat anteriorhorn motoneurons.

Although various biological aspects of MNTF1 have been widely publicizedin scientific journals, the DNA and inferred amino acid sequences of thecloned human MNTF1 gene and its associated receptor had not yet beenmade publicly available by Dr. Chau, nor had these sequences beenconfirmed by peer-review within the field. Moreover, the cloned humanMNTF1, reported by Dr. Chau in 1993, was not in a form which wasamenable to being sub-cloned into an appropriate vector, such as an invitro mammalian expression system. Thus, there remained a need for thehuman MNTF1 gene to be properly manipulated, sequenced, sub-cloned intoan appropriate vector(s), sub-cloned into an appropriate expressionsystem(s) and associated host(s), as well as the isolation andpurification of the resulting recombinant human MNTF1 protein forsubsequent potential utilization in human therapeutic modalities.

SUMMARY OF THE INVENTION

The present invention is directed to a family of motoneuronotrophicfactors including MNTF1 and MNTF2, which have been shown to havediagnostic and therapeutic applications in mammals. The presentinvention is also directed to novel DNA sequences which encodemotoneuronotrophic factors, including recombinant human MNTF1-F3 [SEQ IDNO:1] and MNTF1-F6 [SEQ ID NO:2], to vectors which contain these novelDNA sequences, to expression systems and associated hosts which containthese novel DNA sequences, and the novel recombinant human MNTF1-F3 [SEQID NO:3] and MNTF1-F6 [SEQ ID NO:4] proteins which are produced by theaforementioned expression systems.

The present invention is also directed to the use of motoneuronotrophicfactors for promoting axonal regeneration, for inhibiting the effects ofhereditary motoneuron disease, for minimizing or inhibiting the effectsof scar tissue formation, and for accelerating wound healing whileconcomitantly minimizing or inhibiting the effects of scar tissue andkeloid formation.

DESCRIPTION OF THE FIGURES

The present invention may be better understood and its advantagesappreciated by those individuals skilled in the relevant art byreferring to the accompanying figures wherein:

FIG. 1A depicts the DNA sequence of the MNTF1-1443 DNA fragment [SEQ IDNO:1] which encodes the MNTF1-F3 protein. By standard convention, theDNA sequence is shown in the 5′ to 3′ directed.

FIG. 1B depicts the DNA sequence of the MNTF1-927 DNA fragment [SEQ IDNO:2] which encodes the MNTF1-F6 protein. The first 99 of thesenucleotides [SEQ ID NO:5] correspond to and code for the 33 amino acidpeptide shown in FIG. 2B. By standard convention, the DNA sequence isshown in a 5′ to 3′ orientation.

FIG. 1C depicts the first 99 nucleotides [SEQ ID NO:5] (33 codons) ofthe DNA sequence shown in FIG. 1B.

FIG. 2A depicts the direct amino acid sequence of MNTF1-F3 protein [SEQID NO:3]. By standard convention, the amino acid sequence is reportedfrom the amino (NH2-) terminus to the carboxyl (—COOH) terminus.

FIG. 2B depicts the direct amino acid sequence of MNTF1-F6 protein [SEQID NO:4] encoded by the MNTF1-927 DNA fragment. By standard convention,the amino acid sequence is reported from the amino (NH2-) terminus tothe carboxyl (—COOH) terminus.

FIGS. 3A-3C depict the “Protein Band-Fishing By Cells” methodology whichshowed that isolated anterior horn motoneurons survived and grew onPhastGel regions containing MNTF1 and MNTF2 from muscle extract whichhad been electrophoretically separated into protein bands within thePhastGel. FIGS. 3A-3C illustrate the results obtained utilizing the“Protein Band-Fishing By Cells” methodology to isolate themotoneuronotrophic factors MNTF1 and MNTF2.

FIG. 4 (panels 1-4) reveals the results of isolated motoneurons culturedwith the 35 kD gel band containing the MNTF1 for 3, 7, and 14 days(panels 2, 3, & 4, respectively) and a control culture (panel 1) withoutthe 35 kD MNTF1-containing gel.

FIG. 5 (panels 1-6) reveals the immunohistochemical distribution ofMNTF1 (panels 1, 3 & 5) and its putative MNTF1 receptor (panels 2, 4 &6) in muscle (panels 1 & 2); Schwann cells (panels 3 & 4); andmotoneurons (panels 5 & 6).

FIG. 6 depicts the axotomy of the sciatic nerve of a 10 day-old SpragueDawley rat. The sciatic nerve at the gluteal region of the right sidewas axotomized with 1 mm of the nerve removed and MNTFs weresubsequently placed around the cut edge of the nerve.

FIGS. 7A and 7B reveal the synergistic effect of MNTF1 (35 kD) and MNTF2(22 kD) on the survival of axotomized motoneurons of the sciatic nervein the Sprague Dawley rats. Panel 1 had no gel added (NG) and panel 3had 22 kD MNTF2 gel added; (I) is the intact side and (A) is theaxotomized side. Panel 2 had the 35 kD MNTF1 gel added and panel 4 hadboth the 35 kD and 22 kD gels added.

FIGS. 8A and 8B provide histogram and tabular results of the synergisticprotection and rescue of axotomized motoneurons of the sciatic nerve bythe partially-purified and isolated motoneuronotrophic factors MNTF1 andMNTF2 following the transection of the sciatic nerve in Sprague Dawleyrats.

FIG. 9 (panels 1-4) reveals the microscopic effects of MNTFs on thesurvival of motoneurons in the facial nuclei of the transected facialnerve in the Sprague Dawley rats. Panel (1) the facial nuclei of thenormal intact control rat after two weeks; panel (2) the axotomizedfacial nuclei without factors after two weeks; panels (3) and (4) theaxotomized facial nuclei with 35 kD MNTF1 and with both 35 kD and 22 kDMNTF1 & MNTF2, respectively, for two weeks.

FIGS. 10A and 10B provide histogram and tabular results of the in vivoeffects of muscle-derived motoneuronotrophic factors (22 kD, 35 kD, and22 kD+35 kD) on neuronal survival following the transection of thefacial nerve in Sprague Dawley rats.

FIGS. 11A and 11B provide histogram and tabular results of the in vivoeffects of muscle-derived 35 kD MNTF1 motoneuronotrophic factor onneuronal survival following the transection of the facial nerve inSprague Dawley rats in the presence of goat anti-rabbit IgG or anti-35kD motoneuronotrophic factor monoclonal antibody.

FIGS. 12A-12D depict a series of four photomicrographs demonstrating thein vivo effects of the 35 kD MNTF1 and 22 kD MNTF2 proteins uponneuronal survival following the transection of dorsal root ganglion inSprague Dawley rats.

FIGS. 13A and 13B show a hemisection of the spinal cord -at the T1-T3level in the adult Sprague Dawley rat depicting the surgicalautographing of the ulnar nerve to the ventral spinal cord and themedian nerve to the dorsal spinal cord.

FIG. 14 (panels 1-6) depicts the trophic and rescuing effects of MNTFson the survival of motoneurons following the hemisection of the spinalcord in Sprague Dawley rat with nerve autographs. Panels 1-4 demonstratethat: (1) with the presence of MNTF1 & MNTF2 there were reduced ininflammation, scar formation, and white blood cells infiltration at thejunction of the autograph nerve (N) and the spinal cord (C); panel (2)with no MNTFs there were increased in scar formation, inflammation, andWBCs infiltration at the junction of the autograph nerve (N) and thespinal cord (C); panel (3) is an electron micrographs of the autographnerve with MNTFs which reveals an increase in number and size of theregenerating nerve fibers with healthy myelination; panel (4) is an EMof the autograph nerve without MNTFs which reveals no healthy myelinatedregenerating nerve fibers.

FIGS. 15A and 15B depict the body weights of homozygous (N1-N4) in panelA and heterozygous (N1-N4) in Panel B wild type mice verses the bodyweights of their litter-mates wobbler mice which were treated with ratMNTFs (W5-W8) and (W1-W4), respectively.

FIG. 16 (panels A-F) depicts the survival (with neurite outgrowth) of invitro isolated anterior motoneuron cultures derived from the lumbarspinal cord following 14 days of co-culture with human recombinant MNTF1(hrMNTF1). Panels a-c isolated motoneuron cultures were photographed atday 2 and panels d-e photographed at day 14; panels a and d representcontrol cultures with no hrMNTF1; and panels b, c, e, and f depictexperimental cultures with hrMNTF1.

FIG. 17 (panels 1-6) depicts the survival of in vitro-isolated anteriormotoneuron cultures derived from the lumbar spinal cord following 8 daysof co-culturing without MNTF1-F6 (panel 1) and MNTF1-F3 (panel 2); withGST-fusion protein of MNTF1-F6 (panel 3) and MNTF1-F3 (panel 4); andwith cleaved protein of MNTF1-F6 (panel 5) and MNTF1-F3 (panel 6). Lowmagnification (×100).

FIG. 18 (panels 1-6) depicts the survival (with neurite outgrowth) of invitro-isolated anterior motoneuron cultures as in FIG. 17 (1-6) above.High magnification (×320).

FIG. 19 (panels 1-2) depicts the survival with neurite outgrowth of invitro-isolated anterior motoneuron cultures following 8 days ofco-culturing with the cleaved protein of MNTF1-F6. High Magnification(×400).

FIGS. 20A-20D depict the survival, growth and regeneration of myelinatednerve fiber from the in vitro-isolated anterior motoneurons from theculture as originally demonstrated in FIGS. 17 (panel 5), 18 (panel 5),and 19 (panels 1-2) after 21 days of co-culturing with the cleavedprotein of MNTF1-F6. FIG. 20A reveals nerve fibers; one ˜1.5 mm and theother 3 mm were regenerated from the cultured motoneurons. Lowmagnification (×40). FIG. 20B is a high magnification (×140) of FIG. 20Aand a composite picture to reveal the myelinated, regenerated nervefiber with oligodendrocytes (possible Schwann cells?*) attached. FIGS.20C and 20D reveal clearly the axon cylinders in the inner regeneratedmyelinated nerve fiber with node of Rannier. High magnification (×400).

DESCRIPTION OF THE INVENTION

The present invention comprises a family of neuronotrophic factors whichpossess the ability to exert a trophic effect on motoneurons and thegenes which encode these factors. These factors have been isolated andthe genes which encode these factors have been cloned and expressed, andboth the nucleic acid and polypeptide sequences provided

It has been demonstrated that the isolated factors and the expressed,recombinant factors are capable of inducing the continued viability andneurite outgrowth of motoneurons. Therefore, these factors have beenclassified as “motoneuronotrophic factors” or “MNTFs.”

MNTFs have been isolated from both rat and human sources and theirbiological activities have been examined both in vivo and in vitro. Forexample, their potential biological activity has been examined in bothsurgically-axotomized and hereditarily diseased animals.

The MNTFs disclosed in the present invention are useful for a variety ofpurposes. They can be utilized in the diagnosis of motoneuron diseasesby exploiting their effectiveness in altering the symptomology of thedisease. For example, a detailed description is provided below whichsupplies evidence of the successful use of MNTF1-F3 and MNTF1-F6 inwobbler mice. Wobbler mice are animals which possess double recessivegenes for a hereditary motoneuron disease. The motoneuron diseasemanifests itself with symptoms of upper limb neuromuscular failure whichinitially appears approximately 3 weeks after birth. The condition alsoaffects the animal's body weight, general health conditions,respiration, and life span in a deleterious manner. The diseasegradually progresses to the final, terminal stage (defined as stage 4)by 3 months of age, with an associated dramatic increase in animalmortality. The symptomatic responsiveness of the wobbler mice to theaforementioned MNTFs is indicative of the defect occurring at themotoneuron level, thus serving to confirm various genetic evidence whichhas established the hereditary nature of the defect.

In addition, MNTFs may also be utilized therapeutically to treat damagedor diseased motoneurons. For example, a detailed description is providedbelow which includes experiments using these factors onsurgically-transected motoneurons, with the resulting recovery of alarge percentage of the transected motoneurons. Also described is theutilization of the factors in the aforementioned hereditary motoneurondisease, the wobbler mouse, with results demonstrating that the treatedanimals survived, with a concomitant arresting of pathologicalsymptomologies, and thrived for much longer periods of time than thoseof the untreated animals.

“Protien Band-fishing by Cells” Methodology

Rat MNTF1 and MNTF2 were isolated utilizing the “Protein Band-Fishing byCells” methodology as reported in Chau, R. M. W., et al., MuscleNeuronotrophic Factors Specific for Anterior Horn Motoneurons of RatSpinal Cord. In: Recent Advances in Cellular and Molecular Biology, Vol.5, Peeters Press, Leuven, Belgium, pp. 89-94 (1992). Trophic factors aregenerally found in minute quantities in vivo; hence this can potentiallycause tremendous difficulties in their isolation utilizing traditionalbiochemical methodologies. In view of this fact, a novel techniquedesignated “Protein Band-Fishing by Cells” was developed in which viableanterior horn motoneurons were co-cultured on an electrophoretic gelcontaining the separated proteins from rat peroneal muscle. Thismethodology thus allowed the viable anterior horn motoneurons to“fish-out” those peroneal muscle proteins which exhibited biologicalactivity specific for those motoneurons (i.e., by the demonstration ofcontinued viability and growth in vitro).

For electrophoretic protein separation, the peroneal longus and brevisskeletal muscles, without the associated tendon, from 3 week-old SpragueDawley rats were aseptically dissected into small sections and washed3-times in Ca⁺²/Mg⁺²-free Hank's medium. The muscle tissue was thenhomogenized in 10 mM Tris-HCl (pH 7.2). A cell lysate was obtained bycentrifugation at 100,000×g. for 10 minutes, and the supernatant wasfiltered utilizing a 0.2 m Millipore filter membrane (Millipore Corp.,MA). The resultant filtrate was designated muscle extract (Me).

For cell culture, the spinal cords from four 3 week-old rats wereexcised and the meninges and any associated vessels were removed. Itshould be noted that the motoneurons were pre-labeled with rhodamine Bfor subsequent microscopic visualization by implantation of gelfoamscontaining rhodamine B into the peroneal muscles of the experimentalanimals one week prior to their sacrifice. The gray matter of theanterior horn region of segments L4-L5 was subsequently removed fromeach excised spinal cord, washed several times in Ca⁺²/Mg⁺²-free medium,and treated with collagenase (0.08%) at 37° C. for 60 minuets. Followinggentle dissociation and sedimentation, the cell were suspended in RPMI1640 medium/6% fetal calf serum/20 mM HEPES/1× streptomycin andpenicillin and utilized for subsequent PhastGel culture.

The filtered, muscle extract (Me, 10-20 1 of a 1 mg/ml solution) wasthen applied to a pre-cast 20% native PhastGel (50×40×0.45 mm, PharmaciaLKB Biotech AB, Upsala, Sweden) for separation by the Phast System gelelectrophoresis (Pharmacia LKB Biotech AB, Upsala, Sweden). Theelectrophoretic conditions utilized were those suggested by the computerprogram of Olsson, I., et al., Computer Program for OptimizingElectrophoretic Protein Separation, 9 Electrophoresis 16 (1988).

Following electrophoresis, the middle portion of the PhastGel wasremoved via aseptic technique for subsequent culture with ˜1×10⁶ spinalcord cells/gel. The remaining portions of the PhastGel were stainedusing the silver stain method to identify and locate the resultingprotein bands. Following 2-7 days of incubation, the PhastGel/spinalcell culture was fixed in 0.4% paraformaldehyde in Phosphate-bufferedsaline (PBS) for approximately 2 hours. This portion of the gel was thenreinserted back into its original position of the PhastGel and examinedfor viable, large (˜25 mm) rhodamine B retrograde-prelabeled motoneuronsin the cultured spinal cells using either an inverted phase (ZeissAxiophot Inverted Microscope, West Germany) or a fluorescent microscope(Zeiss, West Germany).

Silver staining demonstrated a total of 34 native protein bands (26protein bands with an apparent molecular weight of >30 kD and 8 proteinbands with an apparent molecular weight of <30 kD) separated from themuscle extract. These results are illustrated in FIGS. 3A-3C.Interestingly, large (˜25 mm) rhodamine B retrograde-prelabeledmotoneurons from the cultured spinal cells were found to survive in onlytwo spatially-distinct regions corresponding to the 35 kD and 22 kDapparent molecular weight protein bands. These results are demonstratedin FIG. 3B. Moreover, silver staining methodology demonstrated heavierstaining of the 35 kD protein band, in comparison to the 22 kD proteinband, thus potentially reflecting the relative concentrations of thesetwo trophic factors in the muscle extract. No other types of neuronalcells were found to have survived, nor did the aforementionedmotoneurons remain viable in any other locations corresponding toadditional proteins with differing apparent molecular weights. Theseaforementioned results are indicative of the large, rhodamine Bretrograde-prelabeled motoneurons having “fished out,” from the amongstthe 34 total peroneal muscle protein bands, their own trophic factorshaving apparent molecular weights of 35 kD and 22 kD, and areillustrated in FIG. 3C. The 35 kD and 22 kD protein bands weredesignated MNTF1 and MNTF2, respectively.

Determination of MNTF1 and MNTF2 Biological Activity

The rat MNTF1 and MNTF2 proteins were then tested for potentialbiological activity with anterior horn motoneurons. Anterior hornmotoneurons were isolated from four 3 week-old Sprague Dawley rat lumbarspinal cord via collection of the associated gray matter. The collectedgray matter was then digested with a 0.08% collagenase solution in DMEMmedium. The motoneurons in the DMEM medium were collected in 5 mlaliquots and allowed to settle at room temperature for 2 minutes. Theupper 4.5 ml of supernatant was aspirated and discarded and theremaining media, containing the motoneurons, were subsequently utilizedto test the biological activity of the rat MNTF1 and MNTF2 proteins.

Rat MNTF1 and MNTF2 were isolated utilizing the aforementioned PhastSystem gel electrophoresis apparatus by aseptic excision of the PhastGelsections (˜1×30 mm) containing the 35 kD and 22 kD protein bands,respectively (see FIGS. 3A-3C). Anterior horn motoneurons, isolated fromthe lumbar spine of 3 week-old Sprague Dawley rats, were thenco-cultured with and without the presence of MNTF1- and MNTF2-containingPhastGel sections. Results indicated that both MNTF1- andMNTF2-containing PhastGel sections were capable of supporting thecontinued viability of the anterior horn motoneurons, as well assupporting neurite outgrowth.

In a similar experiment, depicted in FIG. 4, panels 1-4, anterior hornmotoneurons, isolated from the lumbar spine of 3 week-old Sprague Dawleyrats, were then co-cultured with and without the presence ofMNTF1-containing PhastGel sections for 3, 7, and 14 days (panels 2, 3, &4, respectively). The results of this experiment are shown in FIG. 4which demonstrates that MNTF1 is capable of supporting the continuedviability of anterior horn motoneurons for 2 or more weeks. Thedetermination of motoneuron viability and neurite outgrowth wasperformed utilizing both MTT micro-assay and microscopic morphologicalmeasurement. MTT is a highly sensitive, calorimetric tetrazoliumderivative [3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazoliumbromide] which can be converted by viable cells to form a formazanproduct which can subsequently be measured with an ELISA calorimeter.The control assay (panel 1), utilizing selected remainingprotein-containing PhastGel sections or PhastGel sections alone, failedto support any continued viability or neurite outgrowth of the anteriorhorn motoneurons.

In Vivo Testing of Rat MNTF1 and MNTF2 in Axotomized Animals

In vivo testing of rat MNTF1 and MNTF2 were next performed utilizingaxotomized animals. Axotomized rat sciatic, facial, hypoglossal, andmusculocutaneous nerves were cut in the right side of 10 day-old SpragueDawley rats. The aforementioned nerves were left intact on the left sideof the rats to serve as contralateral internal controls. Sections ofMNTF1- and/or MNTF2-containing PhastGel (˜2×3 mm sections containing5-30 ng of MNTFs) were applied via implantation to the axotomized sites.The axotomy of the sciatic nerve and subsequent implantation ofMNTF-containing gels(s) are illustrated in FIG. 6. In the surgicalprocedure, the right-side of the gluteal region was dissected so as toreveal the sciatic nerve. A 1 mm section of the sciatic nerve wasresected and the remaining ends of the nerve were spatially alignedwithout performing an end-to-end anastomosis. The MNTF-containing andcontrol gel sections were then placed around the surgical resection siteand the initial incision was closed. Axotomized animals in whichPhastGel without MNTFs were implanted in an analogous manner and wereutilized as controls. After periods of 2, 4, and 8 weeks, the axotomizedanimals were sacrificed and tissue sections were excised and preparedfor determination of motoneuron survival/viability via microscopicexamination.

The results demonstrated that without the application of the MNTFs,60-70% of the motoneurons associated with the aforementioned nerves haddegenerated, therefore only 30-40% of the motoneurons were still viableat the time of microscopic examination. Conversely, in the presence ofeither MNTF1 or MNTF2, 55-75% of the motoneurons displayed continuedviability during the above periods of observation. In the presence ofboth MNTF1 and MNTF2, ˜90% of the motoneurons were found to exhibitcontinued viability.

FIGS. 7 and 8 depict the results of the in vivo effects ofmuscle-derived motoneuronotrophic factors on neuronal survival followingthe transection of the sciatic nerve. As previously discussed, thesciatic nerves were cut in the right side of 10 day-old Sprague Dawleyrats (see FIG. 6); whereas the sciatic nerves were left intact on theleft side of the rats to serve as contralateral internal controls.Section of MNTF1-containing and/or MNTF2-containing PhastGel (˜2×3 mmsection containing 5-30 ng of MNTFs) were applied via implantation tothe axotomized sites. MNTF1 and MNTF2 correspond to the 35 kD and 22 kDproteins, respectively. PhastGel sections without MNTFs were implantedin axotomized animals in an analogous manner, and served as controls.After 3 weeks, the axotomized animals were sacrificed and sections ofthe lumbar spinal cord were excised and prepared for determination ofmotoneuron survival/viability via microscopic examination. Resultsdemonstrated that without the application of the MNTFs, 56% of themotoneurons associated with the aforementioned nerves had degenerated.Therefore, only 44% of the motoneurons were still viable at the time ofmicroscopic examination. Conversely, in the presence of either the 35 kDprotein MNTF1 or the 22 kD protein MNTF2, 76% and 71% of the motoneuronsdisplayed continued viability, respectively, at the time of microscopicevaluation.

FIGS. 7A and 7B represents a series of eight photomicrographs depictingthe in vivo effects of muscle-derived motoneuronotrophic factor onneuronal survival following transection of the sciatic nerve in SpragueDawley rats. These results are presented in tabular and histogram formin FIGS. 8A and 8B, respectively. In this series of experiments,axotomized sciatic nerves were cut in the right side of adult SpragueDawley rats, whereas the left side sciatic nerve was left intact as aninternal contralateral control. Sections of MNTF1-containing orMNTF2-containing PhastGel (˜2×3 mm section containing 5-30 ng of MNTFs)were applied via implantation to the axotomized sites. PhastGel sectionswithout MNTFs were implanted in axotomized animals in an analogousmanner, and served as controls. It should be noted that all the lumbar 4and 5 anterior horn motoneurons were retrogradely prelabeled withhorseradish peroxidase (HRP) for 2 weeks prior to animal sacrifice, atwhich time sections of the sciatic nerve were excised and prepared forsubsequent determination of motoneuron survival/viability via amicroscopic cell counting methodology.

Results shown within the control photomicrographs (see FIG. 7A, panel 1)demonstrated that without the application of the MNTFs (NG), acomparatively low number of viable (i.e., HRP-labeled) motoneurons arepresent in all the serial sections of the axotomized side (A) of thelumbar spinal cord as compared to the intact side. This finding iscorroborated by the tabular results presented in FIG. 8 which show that56% of the HRP-labeled motoneurons associated with the sciatic nerve haddegenerated. Conversely, in the presence of the 22 kD protein MNTF2 (seeFIG. 7A, panel 3), the 35 kD protein MNTF1 (see FIG. 7B, panel 2), orboth the 22 kD and 35 kD MNTFs (see FIG. 7B, panel 4) a markedly higherrate of survival of the HRP-labeled motoneurons was demonstrated. Theseresults are quantitatively depicted in FIGS. 8A and 8B, and arecorroborated by the fact that the utilization of the 35 kD MNTF1 proteinor the 22 kD MNTF2 protein resulted in 76.5% and 71.3% of theHRP-labeled motoneurons displaying continued viability, respectively.Similarly, in the presence of both MNTF1 and MNTF2 (see FIG. 7B, panel4), a large number of viable motoneurons are found with 86.8% of themotoneurons exhibiting continued viability as a result of treatment withboth MNTFs (see FIGS. 8A and 8B).

FIG. 9, panels 1-4 illustrates the results of the in vivo effects ofMNTF application on neuronal survival and viability following thetransection of the rat facial nerve in photomicrographic form. In thisseries of experiments, axotomized facial nerves were cut in the rightside of 10 day-old Sprague Dawley rats, whereas the left-side facialnerves were left intact to serve as an internal contralateral control.Sections of MNTF1-containing or MNTF2-containing PhastGel (˜2×3 mmsection containing 5-30 ng of MNTFs) were applied via surgicalimplantation to the axotomized sites. As before, the MNTF1 and MNTF2correspond to the 35 kD and 22 kD proteins, respectively. PhastGelsections without MNTFs were implanted in axotomized animals in ananalogous manner, and served as controls. After periods of 1 and 2weeks, the axotomized animals were sacrificed and serial sections of thefacial nuclei in the brain were prepared for subsequent evaluation ofmotoneuron survival/viability via microscopic examination followinghistological staining. Results in FIG. 9, panel 1 depicts the facialnuclei of the normal intact control rat after two weeks; panel 2 depictsthe axotomized facial nuclei without factors after two weeks; panels 3and 4 depict the axotomized fazial nuclei with 35 kD MNTF1 and with both35 kD and 22 kD MNTF1 & MNTF2, respectively, after two weeks. FIG. 10Aand 10B illustrate the results in tabular and histogram form,respectively. The results shown in FIGS. 9, 10A and 10B demonstratedthat without the application of either the MNTFs, ˜50% of themotoneurons associated with the facial nuclei had degenerated after onlyone week and ˜65% had degenerated after two weeks (panel 1). Incontrast, in the presence of either the 35 kD protein MNTF1 (panel 3) orthe 22 kD protein MNTF2 (panel 2), ˜70% of the motoneurons displayedcontinued viability after 1 week and ˜55% were still viable after 2weeks. Similar results were obtained when both the 35 kD (MNTF1) and 22kD (MNTF2) proteins were utilized (panel 4).

FIGS. 11A and 11B depict the results of the in vivo effect ofmuscle-derived motoneuronotrophic factors on neuronal survival andviability following the transection of the rat facial nerve in thepresence of either goat anti-rabbit IgG or an anti-35 kD monoclonalantibody in tabular and histogram form, respectively. The development ofthe anti-35 kD MNTF1 monoclonal antibody will be discussed in asubsequent section herein. The goat anti-rabbit IgG was utilized as acontrol to ensure that there was no inhibition of the biologicalactivity of the 35 kD MNTF1 protein due to non-specific interactions.Results indicated that 80% of the motoneurons associated with the facialnerve were still viable after a 2 week period. In contrast, a markedreduction in motoneuron viability (50% viability after 2 weeks) wasfound when an anti-35 kD MNTF1 monoclonal antibody was utilized.Therefore, the diminution of neuronal viability appeared to be afunction of the inhibition of the 35 kD MNTF1 protein by the anti-35 kDMNTF1 monoclonal antibody.

FIGS. 12A-12D are a series of four photomicrographs depicting the invivo effects of MNTFs on neuronal survival and viability following thetransection of the dorsal root ganglion in Sprague Dawley rats. In thisseries of experiments, axotomized dorsal root ganglion were cut in theright side of 10 day-old Sprague Dawley rats. The left-side dorsal rootganglion were left intact to serve as an internal contralateral control.Sections of MNTF1-containing or MNTF2-containing PhastGel (˜2×3 mmsection containing 5-30 ng of MNTFs) were applied via implantation tothe axotomized sites. As before, the MNTF1 and MNTF2 correspond to the35 kD and 22 kD proteins, respectively. PhastGel sections without MNTFswere implanted in axotomized animals in an analogous manner, and servedas controls. Results demonstrated that without the presence of eitherMNTF1 or MNTF2, only ˜40% of the motoneurons remained viable [see FIG.12A]. In contrast, 60-70% remained viable in the presence of MNTF1 [seeFIG. 12B], MNTF2 [see FIG. 12C] or both MNTF1 and MNTF2 [see FIG. 12D].

In Vivo Testing of MNTF1 and MNTF2 in Hemi-sectioned Spinal Cords withNerve Autographs

In the next series of experiments, depicted in FIGS. 13A and 13B andFIG. 14 (panels 1-6), rat MNTF1 and MNTF2 were tested in vivo inhemi-sectioned spinal cord rats with peripheral nerve autographs.Following anesthesia with barbitol, the left side of the spinal cord ofadult Sprague Dawley rats were hemi-sectioned at the T1-T3 (8 mm) level.Under a dissection microscope, 20 mm long ulnar and median nerve trunksfrom the left fore limb were dissected and grafted onto the T1-T3 regionof the spinal cord at the lateral-ventral and lateral-dorsal portions,respectively [see FIG. 13B]. The nerve grafts were anchored to thespinal cord by suturing their associated membranes together with 10-0thread. In the experimental group, a total of 16 sections (˜1×1 mm) ofMNTF1-containing and MNTF2-containing PhastGel were placed in closeproximity to the junctions of the nerve grafts and spinal cord. In acontrol group, PhastGel sections without MNTFs were implanted in ananalogous manner.

Results revealed that, in the presence of the MNTF1-containing andMNTF2-containing PhastGel sections [see FIG. 14, panels 1 and 3,respectively], the survival rate, the overall recovery rate, the numbersof both regenerated myelinated and unmyelinated nerve fibers, and thenumber of surviving motoneurons of the hemi-sectioned rats [see FIG. 14,panel 3] were far greater than those exhibited by the control groupwithout the presence of MNTFs [see FIG. 14, panels 2 and 4]. The resultswere obtained by both general and histological observations which wererecorded and compared. With respect to general observations, the rat'sambulatory ability (i.e., the ability to crawl or walk) and reaction todigit compression were recorded. Histological observations includeddetermination of inflammatory response (presence of infiltratingmacrophages, lymphocytes, and scar tissue), morphology andultrastructure of neurons, the presence of myelinating Schwann cells,and the presence of myelinated nerves. The results demonstrated that theexperimental animals treated with MNTFs recovered both motor and sensoryneuronal function in a far more efficacious manner than the controlanimal group. The experimental animal group also exhibited minimalinflammatory response, little or no scar tissue formation, normalSchwann cell morphology, and normal myelinated and unmyelinated nervemorphology [see FIG. 14, panel 3]. In contrast, the control animal groupexhibited an inflammatory response, indicated by large numbers ofinfiltrating macrophages/lymphocytes and collagen-containing scar tissueformation at the location of the neuronal graft [see FIG. 14, panel 5].Furthermore, the Schwann cells of the control animal group were eithernon-viable or exhibited a swollen morphology with vacuolations [see FIG.14, panel 2].

In addition, subsequent experimental results demonstrated that theapplication of MNTF1 and/or MNTF2 significantly increased the amount,size, and neuronal shape of the regenerated myelinated nerve fiber. Fromthese results it may be postulated that MNTFs play a role in thebiosynthesis of the various protein constituents of myelin (e.g., myelinbasic proteins, Wolfgram proteins, etc.). Decreases in theconcentrations of these proteins is implicated in several degenerativediseases of myelinated nerve fibers.

In Vivo Testing of Rat MNTF1 and MNTF2 in Hereditary Motoneuron Disease

The molecular mechanism for human motoneuron diseases, includingAmotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophies (SMA)have not yet been elucidated. However, the wobbler mouse (genotypewr/wr) has been developed and is widely used as an animal model forstudies involving spinal and brainstem motoneuron diseases. See e.g.,Duchen, L. W. and Strich, S. J., An Hereditary Motor Neuron Disease withProgressive Denervation of Muscle in the Mouse: The Mutant “Wobbler,” 31J. Neurol. Neurosurg. Psychiatry, 535 (1968); La Vail, J. H., et al.,Motoneuron Loss in the Abducens Nucleus of Wobbler Mice, 404 Brain Res.127 (1987), whose disclosures are incorporated herein by reference. Thewobbler mouse carries an inherited autosomal double-recessive genemutation in Chromosome 11 (specifically located between the egfr and relgenes), which leads to the progressive degeneration of spinal andbrainstem motoneurons. See Kaupman, K., et al., 13 Genomics 35 (1992),whose disclosure is incorporated herein by reference.

Approximately 3 week postpartum, wobbler mice begin to develop the“wobbling” symptomology (stage 1) with concomitant degeneration ofcervical motoneurons leading to both the wasting of the muscles of theforelimbs and an inability to extend the digits and claws. By 3 monthsof age, the pathologic symptomology will progress to stage 4, with a“clumping together” of all associated joints in the forelimbs (e.g., thewrist, elbow, and shoulder joints), as well as an extensive loss of bodyweight and chronic fatigue. Frequently, however, most wobbler mice dieprior to reaching 3 months of age.

The wobbler mouse model has been utilized in demonstrating the limitedprotective effect of several trophic factors on the degeneration ofmotoneurons including: (i) the Ciliary Neurotrophic Factor (CNTF; seee.g., Sendner, M., et al., Ciliary Motoneurotrophic Factor Prevents theDegeneration of Motor Neurons After Axotomy, 345 Nature 440 (1990)),(ii) the Brain-Derived Neurotrophic Factor (BDNF; see e.g., Oppenheim,R. W., et al., Brain-Derived Motoneurotrophic Factor Rescues DevelopingAvian Motoneurons From Cell Death, 360 Nature 755 (1992)), (iii) theGanglion-Derived Neurotrophic Factor (GDNF; see e.g., Henderson, C. E.,et al., GDNF: A Potent Survival Factor for Motoneurons Present inPeripheral Nerve and Muscle, 266 Science 1062 (1994), and (iv) thecombination of CNTF and BDNF (see e.g., Mitsumoto, I. L., et al., 265Science 1107 (1994)). In these aforementioned studies, the treatedwobbler mice were administered with milligram (mg) concentrations of thecorresponding trophic factor at each dosing, with a treatment scheduleconsisting of several dosings per week for a period of a number ofweeks. In most cases, response by the wobbler mice to treatment with thetrophic factor (as measured by improvement of motor function) werescored during a one month time period. Interestingly, however, thetrophic factor-treated mice frequently died sooner than the controlwobbler mice which did not receive treatment. See e.g., Ikeda, S., etal., Histometic Effects of Cilliary Neurotrophic Factor in Wobbler MouseMotor Neuron Disease, 37 Ann. Neurol. 47 (1995); Ikeda, S., et al.,Effects of Brain-Derived Neurotrophic Factor on Motor Dysfunction inWobbler Mouse Motor Neuron Disease, 37 Ann. Neurol. 505 (1995).

The “negative” results demonstrated by these aforementioned trophicfactor studies raise the question of whether these investigators wereworking with the appropriate trophic factor(s) and/or the appropriatebiological assays. Therefore, based upon the documented relationshipbetween motoneurons and their associated target muscles, the inventor ofthe invention disclosed herein chose to isolate and purify themotoneuronotrophic factors (MNTF1 and MNTF2) identified by themotoneurons themselves from the target tissue of the respectivemotoneuron (i.e., the skeletal muscle); and to study the survival curveof the treated wobbler mice after only a single dose of MNTF1administered at nanogram (ng) concentrations for a period of one year.It should be noted that the MNTF concentration utilized in this studywas 1000-times less (i.e., ng versus mg concentrations) than thatutilized in previous studies involving several other motoneuronotrophicfactors (e.g., See e.g., Ikeda, S., et al., Histometic Effects ofCilliary Neurotrophic Factor in Wobbler Mouse Motor Neuron Disease, 37Ann. Neurol. 47 (1995); Ikeda, S., et al., Effects of Brain-DerivedNeurotrophic Factor on Motor Dysfunction in Wobbler Mouse Motor NeuronDisease, 37 Ann. Neurol. 505 (1995)).

In a series of experiments, depicted in FIGS. 15A and 15B, in vivotesting of rat MNTF1 and MNTF2 in hereditary motoneuron disease wasperformed in wobbler mice. Wobbler mice are animals (developed at andkindly provided by the National Institutes of Health (NIH) in Bethesda,Md.) which possess double recessive genes (wr/wr) for a hereditarymotoneuron disease. The motoneuron disease manifests itself withsymptoms of upper limb neuromuscular failure which initially appearsapproximately 3 weeks after birth. The condition also affects theanimal's body weight, general health conditions, respiration, and lifespan in a deleterious manner. The disease gradually progresses to thefinal stage (stage 4) by 3 months of age, with an associated dramaticincrease in animal mortality.

Sections (˜2×6 mm) of MNTFl-containing and MNTF2-containing PhastGelwere finely minced and applied to the wobbler mice via implantation ofthe aforementioned MNTF-containing PhastGel sections between thetrapezius and rhomboid muscles at the C7-T3 region of the spinal cord. Acontrol group of wobbler mice received PhastGel sections without MNTFs.Results indicated that the application of a single “dose” (35 mg/kg bodyweight) of MNTF-containing PhastGel to 6 week old wobbler mice arrestedthe progression of the symptomology of the motoneuron disease to thatassociated with the initial stage (stage 1) for a period of observationup to 10 months. Additionally, in comparison to the control animalgroup, the experimental animals demonstrated a general improvement inhealth, respiration, body weight, strength of fore limbs, as well as thegeneral prevention of deterioration of health, respiration,neuromuscular activities of the fore limbs, and the vacuolation andchromatolysis of their cervical motoneurons. In conclusion, theseresults are illustrative of rat MNTFs arresting the further symptomaticdevelopment of hereditary motoneuron disease in wobbler mice.

FIGS. 15A and 15B graphically depicts the weights of homozygous andheterozygous wild type mice verses wobbler mice. In this series ofexperiments, the body weights of four “normal” homozygous mice (A) andfour heterozygous mice (B) were compared with the body weights of foureach homozygous wobbler mice (W1-W4) and (W5-W8). Each of the wobblermice were treated with rat MNTF1 and MNTF2, whereas the “normal” micewere left untreated. While, the overall body weights of the wobbler micewere generally less than that of “normal” animals, the wobbler micenonetheless exhibited consistent weight gain and increased longevity. Incontrast, untreated wobbler mice did not demonstrate this type ofconsistent weight gain and usually die at approximately 3 months of age.It should be noted that the abrupt “drop-points” in both FIGS. 15A and15B represent the date on which that particular animal expired.Therefore, in conclusion, these results indicate that treatment with theMNTFs had a dramatic impact on the hereditary motoneuron disease inwobbler mice.

The elucidation of the relationship between the individual muscle fibersand their associated motorneurons is of paramount importance inunderstanding such diseases as multiple sclerosis (MS), musculardystrophy (MD), and myasthenia gravis (MG). In a related series ofexperiments, it was demonstrated that the application of MNTF1 and/orMNTF2 which resulted in a marked regeneration of the motorneuronsconcomitantly led to the recovery associated muscle fibers formation.Moreover, neuro-stimulation of the muscle fiber to promote the growth,survival, and regeneration of the muscle fiber, as well as for theputative production of muscle-derived MNTFs, plays an extremelyimportant role in the cyclic relationship between motorneurons and themuscle fibers.

Development of Monoclonal Antibodies for MNTF1 and MNTF2

Monoclonal antibodies specific for rat MNTF1 and MNTF2 were prepared.The 35 kD (MNTF1) and 22 kD (MNTF2) protein-containing bands wereexcised from a Phast System gel (1×30 mm gel sections containing ˜100 ngmotoneuronotrophic factor) and utilized as antigens in the immunizationof separate groups of Balb/c mice. Specifically, the MNTF-containingPhastGel sections were excised and finely minced. The PhastGel pieceswere then mixed with an equal volume of complete Freund's adjuvant anddirectly injected intraperitoneally into the Balb/c mice. A total ofthree antigen immunizations were performed, with intraperitonealinjections of MNTF1-containing and MNTF2-containing PhastGel inphysiological saline on the 7th and 21st day following the initialimmunization. The spleens of the Balb/c mice were harvested and allowedto fuse with either NS-1 or SP2/0 myeloma cells. The resultanthybridomas were then screened utilizing MTT microassays and microscopicexamination to select the most efficacious monoclonal antibodies capableof blocking the function of the MNTF1 and MNTF2 neuronotrophic factors.For example, from the 35 kD (MNTF1) fusions, a total of 8 hybridomaswere selected and subsequently developed out of a total of 480 wellsproducing monoclonal antibodies specific for this aforementioned MNTF1protein.

Immunoselection of Recombinant Human MNTF1

The selected MNTF-blocking monoclonal antibodies were next utilized toimmunoselect clones of human motoneuronotrophic factor. The blockingmonoclonal antibody for the human motoneuronotrophic factor (MAb-MNTF)was used as an immunoprobe in the screening of positive expressionclones from a selected human retinoblastoma cDNA library (produced byClontech Co., Palo Alto, Calif.). In brief, theimmunoselection/immunoprecipitation procedure for the cDNA libraryinvolved the isolation of mRNAs from cells of the Rb Y-79 retinoblastomacell line. Reverse transcription reaction were performed to synthesizecDNAs utilizing the isolated retinoblastoma mRNAs as templates. ThecDNAs were ligated into a Lambda phage vector (gt-11) and subsequentlytransformed in an E. coli strain Y1090 host bacteria.

The immunoscreening procedure utilized was a modification of thatdescribed in Young, R. A., and Davis, R. W., Efficient Isolation ofGenes Using Antibody Probes, 80 Proc. Nat'l Acad. Sci. USA 1194 (1983),whose disclosure is incorporated herein by reference. The modificationconsisted of using ammonium nickel sulfate as an enhancing agent toincrease 100-fold the sensitivity of the peroxidase-avidin-biotincomplex reaction utilizing diamino-benzidine as the substrate. A maximumof 4 nitrocellulose membrane “replicas” were made from each colony platefor immunoscreening and only the most intensely-stained clones wereselected in the immunoscreening procedure. The selected clones were thenallowed to express their recombinant human proteins which weresubsequently assayed to determine their potential MNTF biologicalactivity in culture. The clone which displayed the highest MNTF-specificbiological activity in motoneuron cultures was then selected for furtheranalysis.

Immunohistochemical Localization of MNTF1 and the MNTF1 Receptor

The MNTF1 monoclonal antibody was utilized to elucidate theimmunohistochemical distribution of MNTF1 and the putative MNTF1receptor in the peroneal muscle, Schwann cells, and motoneurons of 10day-old Sprague Dawley rats. The procedure utilized was previouslydescribed in Ren, F. & Chau, R. M. W., Production and Assessment ofMonoclonal Antibodies Specific for Rat Retinal Ganglion NeuronotrophicFactor, 7 J. Monoclonal Antibody 13 (1991). FIG. 5 demonstrates theresults of this immunohistochemical localization of the MNTF1 protein(plates 1, 3 and 5) in peroneal muscle (plates 1 and 2), Schwann cells(plates 3 and 4) and motoneurons (plates 5 and 6). The results show anuneven distribution of fine, immunoreactive-positive “staining” amongthe muscle fibers with the more dense staining localized at theT-tubules, the motor end-plates, and the intervening nerve fibers. Thestriations of the individual muscle fibers were clearly visible withoutthe utilization of a counter-stain. In addition FIG. 5 illustrates theimmunohistochemical localization of the putative MNTF1 receptor (plates2, 4, and 6).

Cloning and Sequence Analysis of Human Recombinant MNTF1

Human recombinant MNTF1 (hrMNTF1) was tested in vitro for potentialbiological activity with isolated anterior horn motoneurons in thefollowing manner [see FIG. 16, panels A-F]. Anterior horn motoneuronswere isolated from the lumbar spinal cord gray matter of 10 day-oldSprague Dawley rats following digestion in a 0.08% collagenase solutionin DMEM medium containing 15% fetal calf serum. Following collagenasedigestion, the anterior horn motoneurons were cultured in DMEM mediumsupplemented with 15% fetal calf serum. Results indicated that theanterior horn motoneurons co-cultured with hrMNTF1 remained viable andexhibited higher levels of neurite outgrowth [see FIG. 16, panel E andpanel F] than that of the control cultures without hrMNTF1 [see FIG. 16,panel D]. Viability and neurite outgrowth were determined utilizing bothMTT microassay and microscopic examination as previously described.

The cloned, recombinant human MNTF1 in gt-11 phage vector (designated asLambda.35KD.MNTF1) was expressed in E. coli strain Y1090. It was found,in vitro, to be capable of reducing, by a factor of 3-fold, the overallnumbers of isolated anterior horn motor neurons which entered intoapoptosis (the process of cellular “dying”) as evidenced by a lack ofcellular fragmentation into apoptotic bodies and the condensation ofchromatin in the pyknotic nucleus. In addition, hrMNTF1 supported thegrowth and “spreading” of the motoneurons into giant, active neuronswith extended growth cone-containing axons [see FIG. 16, panels E andpanel F]. In contrast, the control motoneuron cultures, lackingexpressed hrMNTF1, many non-neuronal cells (e.g., glial cells andfibroblasts) were actively growing after 10 days of culture in DMEMmedium supplemented with 15% fetal calf serum [see FIG. 16, panel D],yet there was a complete lack of viable, growing motoneurons in thesecontrol cultures.

The cDNA insert of the Lambda.35KD.MNTF1 clone was then sub-cloned intoan in vitro expression vector system and its DNA sequence was elucidatedby the following methodologies:

1. Preparation and Purification of Lambda.35KD.MNTF1

10 plaque-forming units (pfu) of Lambda.35KD.MNTF1/gt 11 phage wasinoculated into a 500 ml overnight culture of E. coli stain Y1090 untilcomplete lysis of the bacteria was observed. The Lambda phage wererecovered and purified by centrifugation and enzymatic treatment withRNase and DNase in an NaCl/PEG 8000 solution, followed by high speedCsCl density gradient centrifugation to collect the purified phage at afinal gradient density of approximately 1.5 gm CsCl/ml.

Isolation and purification of the Lambda.35KD.MNTF1 DNA was facilitatedby initial digestion of the Lambda phage (1 ml) in EDTA, SDS, andproteinase K, followed by repeated extractions with phenol,phenol/chloroform, and chloroform. The DNA was then ethanol precipitatedwith 95% ethanol and collected by centrifugation. Following repeatedwashes in 70% ethanol, the Lambda.35KD.MNTF1 DNA pellet was redissolvedin Tris-EDTA buffer.

2. Recovery of 35KD.MNTF1 cDNA from Lambda.35KD.MNTF1 DNA

10 g of Lambda.35KD.MNTF1 DNA was digested overnight with EcoR1 and thecleaved 35KD.MNTF1 cDNA was recovered via agarose gel electrophoresis.Following electrophoresis, the DNA bands were visualized utilizing U.V.light. Results of the EcoR1 digestion demonstrated the presence of twodiscreet DNA fragments—a 1.44 Kbp fragment designated 35F3 and a 0.93Kbp fragment designated 35F6. The two DNA bands were individuallyexcised from the agarose gel and the DNA was recovered via standardtechniques. The recovered DNA was then prepared for subsequent DNAsequencing by recombination with M13 phage and for High ProteinExpression by recombination with the pGEX-1 Lambda TEcoR1/BAP vector.

3. DNA sequencing of the 35F3 and 35F6 DNA fragments of 35KD.MNTF1 cDNA

Following recombination of the 35F3 and 35F6 fragments with M13, theDideoxy Nucleotide Chain Termination DNA Sequencing Methodology ofSanger (utilizing Bst DNA polymerase) was employed to elucidate the DNAsequence of the two MNTF1 DNA fragments. See Sanger, F., et al.,Nucleotide Sequence of Bacteriophage DNA, 162 J. Mol. Biol. 729 (1982),whose disclosure is incorporated herein by reference. This sequencingmethodology provided extremely accurate and reproducible results withrespect to the DNA sequencing of the two EcoR1-generated fragments ofthe 35KD.MNTF1 cDNA fragments and indicated that the 35F3 fragmentconsisted of 1443 base pairs; whereas the 35F6 fragment was found toconsist of 927 base pairs.

FIG. 1A depicts the DNA sequence (SEQ ID NO:1) of the 35F3 DNA fragment(1443 bp). By standard convention the DNA sequence is shown in the 5′ to3′ orientation. The 35F3 clone was constructed by EcoR1 digestion of theLambda 35KD.MNTF1 clone. The resulting 35F3 EcoR1-generated fragment wasthen recombined with M13 for subsequent DNA sequencing utilizing theaforementioned Sanger methodology.

FIG. 1B depicts the DNA sequence (SEQ ID NO:2) of the 35F6 DNA fragment(927 bp). By standard convention the DNA sequence is shown in the 5′ to3′ orientation. The 35F6 clone was constructed by EcoR1 digestion of theLambda 35KD.MNTF1 clone. The resulting 35F6 EcoR1-generated fragment wasthen recombined with M13 for subsequent DNA sequencing utilizing theaforementioned Sanger methodology.

4. Sub-cloning of the 35F3 and 35F6 EcoR1-generated MNTF1 DNA Fragments

The 35F3 (1443 base pairs) and 35F6 (927 base pairs) EcoR1-generatedMNTF1 DNA fragments were sub-cloned into the pGEX-1 Lambda T EcoR1/BAPHigh Protein Expression vector [hereinafter pGEX]. The resultantsub-clones were designated MNTF1-1443 (35F3) and MNTF1-927 (35F6). ThepGEX vector was selected due to the following factors: (i) it possesseda high efficiency transcriptional promotor at its 5′-terminus and a3′-terminus poly(A) tail; and (ii) it provided an easy methodology forthe purification of the MNTF1-1443 and MNTF1-927 recombinant proteinsvia affinity column chromatography-based purification of theglutathione-S-transferase (GST)-containing MNTF1 fusion proteins.

A. Transformation of recombinant plasmid

The pGEX vector was digested with EcoR1. The digested pGEX vector wasthen incubated overnight with the EcoR1-generated MNTF1-1443 andMNTF1-927 DNA fragments in the presence of T4 DNA ligase. Followingligation, competent E. coli strain DH5 were transformed with therecombinant vector and transferred onto Luria broth (LB) agar platescontaining 100 g/ml ampicillin. Due to the fact that the pGEX vectorcontained a gene which conferred ampicillin resistance to thetransformed bacteria, the use of ampicillin screening allowed theexclusive selection of transformed bacterial colonies, as only thosebacteria containing the recombinant vector would be viable in itspresence.

B. Identification and isolation of MNTF1-1443 and MNTF1-927transformants

The transformed bacterial colonies were individually selected,inoculated into a small volume of LB medium containing 100 g/mlampicillin, and incubated for approximately 3 hours. Followingincubation, the transformed bacteria were collected via centrifugationfor subsequent isolation of the recombinant vector DNA by the alkalinelysis “mini prep” technique as described in Maniatis, T., Fritsch, E.F., and Sambrook, J., Molecular Cloning, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. pp. 134-136 (1986). With thistechnique, the collected bacteria were lysed by the addition of aTris-EDTA/NaOH/SDS solution with vigorous vortexing. Aftercentrifugation to collect contaminating cellular debris, the recombinantvector DNA-containing supernatant was aspirated, extracted with phenoland chloroform, and precipitated with 95% ethanol. The mixture wascentrifuged to collect the precipitated DNA and the nucleic acid pelletwas dissolved in Tris-EDTA buffer. Two recombinant species wereidentified: pGEX-MNTF1-1443 and pGEX-MNTF1-927.

The collected recombinant vector DNA was then digested with EcoR1 torelease the 1443 bp MNTF1-1443 and 927 bp MNTF1-927 DNA inserts from the4.9 Kbp pGEX vector. The digested DNA was subjected to agarose gelelectrophoresis and the individual DNA bands were identified via U.V.light visualization.

“Positive” bacterial colonies (i.e., those which contained either theMNTF1-1443 or MNTF1-927 DNA insert) were selected and inoculated into LBmedium for large scale plasmid purification via alkaline lysis and PEGprecipitation. The purified recombinant vector DNA was then transfectedinto an E. coli strain BL-21 host bacterium to facilitate high levels ofexpression of human recombinant MNTF1-F3 (hrMNTFl-F3) and MNTF1-F6(hrMNTF1-F6) proteins.

C. Expression and Amplification of hrMNTF1-F3 and hrMNTF1-F6 FusionProteins

pGEX-MNTF1-1443 or pGEX-MNTF1-927 DNA were transfected into E. colistain BL-21 competent bacteria in 50 ml of LB medium. IPTG was utilizedto induce high levels of expression of the hrMNTF1-F3 or hrMNTF1-F6proteins. Collected bacteria were lysed, frozen, and thawed a total of4-times using liquid nitrogen, sonicated, and centrifuged. Thesupernatant, containing the hrMNTF1-F3 or hrMNTF1-F6 fusion proteins,was then passed through an anti-GST affinity chromatography columncontaining anti-GST monoclonal antibodies (Pharmacia). The use of thistype of affinity chromatography allowed purification of theGST-hrMNTF1-F3 and GST-hrMNTF1-F6 fusion proteins which were bound tothe matrix through the GST moiety. Following a high salt wash, theGST-hrMNTF1-F3 and GST-hrMNTF1-F6 fusion proteins were eluted. Theproteolytic enzyme thrombin was utilized to cleave the linkage betweenthe MNTFs and the GST moiety and the purified hrMNTF1-F3 and hrMNTF1-F6proteins were collected for subsequent in vivo and in vitro assays andfor amino acid sequencing.

In Vitro and In Vivo Testing of the Trophic Activity of hrMNTF1-F3 andhrMNTF1-F6

The human recombinant motoneuronotrophic factors (hrMNTFs) may beutilized either as fusion proteins or as purified proteins. To obtainthe purified protein form of hrMNTF1-F3 and MNTF1-F6, the expressionproducts of the pGEX-MNTF1-1443 or pGEX-MNTF1-927 plasmids are isolatedas previously described. The expression products consist of fusionproteins which contain MNTF fused with glutathione-S-transferase (GST).The fusion protein is then purified on a GST-monoclonal antibodyaffinity chromatography column (Pharmacia) which specifically cleave thebond between the expressed trophic factor (MNTFs) and the GST moiety.The trophic factor is subsequently eluted from the column and may beutilized in the same manner as the purified “native” proteins, asdescribed above.

Anterior horn motoneurons were isolated from the 10 day-old (postnatal)Sprague Dawley rat lumbar spinal cord via collagenase digestion in DMEMmedium supplement with 15% fetal calf serum. FIG. 17 represents lowmagnification (100×) photomicrographs of the aforementioned anteriorhorn motoneurons cultured with varying concentrations of theGST-hrMNTF1-F3 (panel 4), GST-hrMNTF1-F6 (panel 3), and hrMNTF1-F3(panel 6) and hrMNTF-F6 (panel 5) proteins with the GST moiety removedby thrombin proteolysis were added to the motoneuron cultures. Following8 days of culture, it was observed that the motoneurons cultured in thepresence of GST-conjugated or non-GST-conjugated hrMNTF1-F3 orhrMNTF1-F6 exhibited far greater growth patterns, as well as a markeddecrease in the growth of associated non-neuronal cells, than thosemotoneurons cultured without either of these aforementioned proteins[see FIG. 17, panel 1 and 2]. The similarity of the results obtainedbetween motoneuron cultures with hrMNTF1-F3 and hrMNTF1-F6 suggestedthat both pGEX-MNTF1-1443 and pGEX-MNTF1-927 may potentially possess thegene, or part of the gene which encodes the active site of abiologically active neurotropic factor.

FIG. 18 depicts high magnification (320×) photomicrographs of theresults initially illustrated in FIG. 17. It should be noted that panels1-6 in FIG. 18 are identical to those panels shown in FIG. 17 withrespect to the type of MNTF protein which was utilized in the co-culturewith the isolated anterior horn motoneurons.

FIG. 19 represents a high magnification (400×) photomicrograph ofmotoneuron viability and neurite outgrowth of in vitro-isolated anteriorhorn motoneurons cultured with hrMNTF1-F6 which has had the GST moietyremoved by thrombin proteolysis.

FIGS. 20A-20D depict the survival, regeneration and neurite outgrowth ofmyelinated nerve fiber from the in vitro-isolated anterior hornmotoneurons as originally shown in FIG. 17 (plate 5), FIG. 18 (plate 5),and FIG. 19 (plate 1), respectively, after 21 days of co-culture withthe hrMNTF1-F6 protein with the GST moiety removed via thrombinproteolysis. FIG. 20A illustrates a low magnification photomicrograph(100×) of two separate motoneurons (one ˜1.5 mm in length and one ˜3 mmin length) which were regenerated from the cultured, in vitro-isolatedanterior horn motoneurons. FIG. 20B depicts a low magnification (140×)photomicrograph of the motoneurons originally illustrated in FIG. 20A aswell as a composite photomicrograph which illustrates the myelinated,regenerated neuron with oligodendrocytes (putative Schwann cells)attached. FIGS. 20C and 20D illustrate high magnification (400×)photomicrographs which reveal the axonal cylinders in the innerregenerated myelinated nerve fiber with several Nodes of Rannier clearlydepicted.

Amino Acid Sequencing of the Human Recombinant MNTF1-F3 and MNTF1-F6Proteins

The amino acid sequence of the human recombinant MNTF1-F3 and MNTF1-F6proteins were elucidated by direct protein sequencing methodologies.Prior to sequencing, the proteins were purified into non-fusion form viathe aforementioned GST-monoclonal affinity column chromatography. Theamino acid sequence of the MNTF1-F3 protein [SEQ ID NO:3] is shown inFIG. 2A. The amino acid sequence of the MNTF1-F6 protein [SEQ ID NO:4]is shown in FIG. 2B. By standard convention, the amino acid sequencesare reported from the amino (NH2-) terminus to the carboxyl (—COOH)terminus.

Preparation of MNTF For Drug Delivery

The MNTFs of the present invention can thus be readily utilized inpharmacological applications. In vivo applications includeadministration of the factors to mammalian subjects and, in particular,to human subjects. The MNTFs may be administered orally or by injection,with the preferred mode being topical application on or near theaffected motoneuron.

The pharmacological compositions of the present invention are preparedin conventional dosage unit forms by the incorporation of one or more ofthe MNTFs with an inert, non-toxic pharmaceutical “carrier” moietyaccording to accepted methodologies, in a non-toxic concentrationsufficient to produce the desired physiological activity in a mammaland, in particular, a human subject. Preferably, the compositioncontains the active ingredient in a biologically-active, but non-toxic,concentration selected from a concentration of approximately 5 ng to 50mg of active ingredient per dosage unit (e.g., per kg subject bodyweight). The concentration utilized will be dependent upon such factorsas the overall specific biological activity of the ingredient, specificbiological activity desired, as well as the condition and body weight ofthe subject.

The pharmaceutical carrier or vehicle employed may be, for example, asolid or liquid and a variety of pharmaceutical forms may be employed.Thus, when a solid carrier is utilized, the preparation may be plainmilled, micronized in oil, tabulated, placed in a hard gelatin orenterically-coated capsule in micronized powder or pellet form, or inthe form of a troche, lozenge, or suppository. The solid carrier,containing the MNTF, can also be ground up prior to use.

When utilized in a liquid carrier, the preparation may be in the form ofa liquid, such as an ampule, or as an aqueous or non-aqueous liquidsuspension. For topical administration, the active ingredient may beformulated using bland, moisturizing bases, such as ointments or creams.Examples of suitable ointment bases include, but are not limited to,petrolatum plus volatile silicones, lanolin, and water in oil emulsionssuch as Eucerin® (Beiersdorf). Examples of suitable cream bases include,but are limited to, Nivea Cream® (Beiersdorf), cold cream (USP), PurposeCream®(Johnson & Johnson), hydrophilic ointment (USP), and Lubriderm®(Warner-Lambert).

Additionally, with respect to the present invention, the activeingredient may be applied internally at or near the site of the affectedmotoneuron. For example, a solid or gelled medium which is sufficientlypermeable to allow the release of the active ingredient, preferably in atimed-release manner, may be utilized for such internal application.Examples of such gels include, but are not limited to, hydrogels such aspolyacrylamide, agarose, gelatin, alginate, or other polysaccharidegums. Furthermore, the active ingredient may be imbedded in a solidmaterial, such as filter paper, which is capable of absorbing andsubsequently releasing the active ingredient, at the appropriate timeand location.

While embodiments and applications of the present invention have beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it would be apparent to those individualswhom are skilled within the relevant art that many additionalmodifications would be possible without departing from the inventiveconcepts contained herein. The invention, therefore, is not to berestricted in any manner except in the spirit of the appended claims.

5 1 1443 DNA Homo Sapiens 1 cgggcttatt attccactga tgagaacctg atcctttccccactcctggg taacgtctgc 60 ttctccagct cccagtacag catctgcttc acgctgggctcctttgccaa gatctatgcc 120 gacacctttg gtgacattaa ttaccaagaa tttgctaaaagactctgggg tgacatctac 180 ttcaacccta agacgcgaaa gttcaccaaa aaggccccaactagcagctc ccagagaagt 240 ttcgtggagt ttatcttgga gcctctttat aagatcctcgcccaggttgt aggtgacgtg 300 gacaccagcc tcccacggac cctagacgag cttggcatccacctgacgaa ggaggagctg 360 aagctgaaca tccgcccctt gctcaggctg gtctgcaaaaagttctttgg cgagttcaca 420 ggctttgtgg acatgtgtgt gcagcatatc ccttctccaaaggtgggcgc caagcccaag 480 attgagcaca cctacaccgg tggtgtggac tccgacctcggcgaagctat gagtgactgt 540 gaccctgatg gccccctgat gtgccacact actaagatgttcagcacaca tgatggagtc 600 cagtttcacc cctttggccg ggtgctgagt ggcaccattcatgctgggca gcctgtgaag 660 gttctggggg agaactacac cctggaggat gaggaagactccccaatttg ccccgtgggc 720 cgcctttgga tctctgtggc cagctaccac atcgaggtgaaccgtgttcc tgctggcaac 780 tgggttctga ttgaaggtgt tgatcaacca attgtgaagacagcaaccat aaccgaaccc 840 cgaggcaatg aggaggctca gattttccga cccttgaagttcaataccac atctgttatc 900 aagattgctg tggagccagt caacccctca gagctgcccaagatgcttga tggcctgcgc 960 aaggtcaaca agagctatcc atccctcacc accaaggtggaggagtctgg cgagcatgtg 1020 atcctgggca ctggggagct ctacctggac tgtgtgatgcatgatttgcg gaagatgtac 1080 tcagagatag acatcaaggt ggctgaccca gttgtcacgttttgtgagac ggtcgtggaa 1140 acatcctccc tcaagtgctt tgctgaaacg cctaataagaagaacaagat caccatgatt 1200 gctgagcctc ttgagaaggg cctggcagag gacatagagaatgaggtggt ccagattacg 1260 tggaacagga agaagctggg agagttcttc cagaccaagtacgattggga tctgctggct 1320 gcccgttcca tctgggcttt tggccctgat gcgactggccccaacattct ggtggatgat 1380 actctgccct ctgaggtgga caaggctctt cttggttcagtgaaggacag catcgttcaa 1440 ggt 1443 2 927 DNA Homo Sapiens 2 ttggggacattttggggtga cacactgaac tgctggatgc tatcagcatt tagtaggtat 60 gctcgatgtcttgcagaagg acatgatggt cctacacagt aaggaatgga ttacctacaa 120 tattaatagcagcctcccat acacactttt gacacccttc cctaaaggat taatatgctc 180 caaccttcctgtccccacag ttcagtggct ctccctaccc tcaccatgat cggatgaaaa 240 aaaataaggtttcacagctt aagagtgaaa ttctggaatc caactacaag ctcataactg 300 tagcatggaacctggtagta gcataataaa taaattttta gtaagaggct taagaaattt 360 tagcaaaaaaagcactccct ttcttcctcc ctacatatct catatgtttt tcaacacaaa 420 aaattctgtgattttagaga aacttcttac agtactttta agttcaaaac cagatgctca 480 ttacagttcttttaaacacc aaactagtca tctcaaaaat atggctaact ctctggacta 540 aattccataggaaaaattat taatttcaaa atgcctaatt tttgatcaat gctgaagagc 600 caagcaatcatgtcctgctt ctcactcagg gcagagttct caggtcagaa gctccggagt 660 ctgtcagagattaaaatatc atctcaacaa ttcacaagct acttctaagt gttaccctaa 720 attagtcactaatcgtttct cccccaactc tatttcacaa attaaagttt acagaattga 780 caaaaaccaaaccaatgaaa caacccaggc tatttgcagg gggggggaaa gagatacccc 840 aaaagtcaaccctatttaca cgtagttaaa agagtgatcc aacagatatt accctccata 900 aagtacctaaaggcaggagc cggaatt 927 3 481 PRT Homo Sapiens 3 Arg Ala Tyr Tyr Ser ThrAsp Glu Asn Leu Ile Leu Ser Pro Leu Leu 1 5 10 15 Gly Asn Val Cys PheSer Ser Ser Gln Tyr Ser Ile Cys Phe Thr Leu 20 25 30 Gly Ser Phe Ala LysIle Tyr Ala Asp Thr Phe Gly Asp Ile Asn Tyr 35 40 45 Gln Glu Phe Ala LysArg Leu Trp Gly Asp Ile Tyr Phe Asn Pro Lys 50 55 60 Thr Arg Lys Phe ThrLys Lys Ala Pro Thr Ser Ser Ser Gln Arg Ser 65 70 75 80 Phe Val Glu PheIle Leu Glu Pro Leu Tyr Lys Ile Leu Ala Gln Val 85 90 95 Val Gly Asp ValAsp Thr Ser Leu Pro Arg Thr Leu Asp Glu Leu Gly 100 105 110 Ile His LeuThr Lys Glu Glu Leu Lys Leu Asn Ile Arg Pro Leu Leu 115 120 125 Arg LeuVal Cys Lys Lys Phe Phe Gly Glu Phe Thr Gly Phe Val Asp 130 135 140 MetCys Val Gln His Ile Pro Ser Pro Lys Val Gly Ala Lys Pro Lys 145 150 155160 Ile Glu His Thr Tyr Thr Gly Gly Val Asp Ser Asp Leu Gly Glu Ala 165170 175 Met Ser Asp Cys Asp Pro Asp Gly Pro Leu Met Cys His Thr Thr Lys180 185 190 Met Phe Ser Thr His Asp Gly Val Gln Phe His Pro Phe Gly ArgVal 195 200 205 Leu Ser Gly Thr Ile His Ala Gly Gln Pro Val Lys Val LeuGly Glu 210 215 220 Asn Tyr Thr Leu Glu Asp Glu Glu Asp Ser Gln Ile CysThr Val Gly 225 230 235 240 Arg Leu Trp Ile Ser Val Ala Arg Tyr His IleGlu Val Asn Arg Val 245 250 255 Pro Ala Gly Asn Trp Val Leu Ile Glu GlyVal Asp Gln Pro Ile Val 260 265 270 Lys Thr Ala Thr Ile Thr Glu Pro ArgGly Asn Glu Glu Ala Gln Ile 275 280 285 Phe Arg Pro Leu Lys Phe Asn ThrThr Ser Val Ile Lys Ile Ala Val 290 295 300 Glu Pro Val Asn Pro Ser GluLeu Pro Lys Met Leu Asp Gly Leu Arg 305 310 315 320 Lys Val Asn Lys SerTyr Pro Ser Leu Thr Thr Lys Val Glu Glu Ser 325 330 335 Gly Glu His ValIle Leu Gly Thr Gly Glu Leu Tyr Leu Asp Cys Val 340 345 350 Met His AspLeu Arg Lys Met Tyr Ser Glu Ile Asp Ile Lys Val Ala 355 360 365 Asp ProVal Val Thr Phe Cys Glu Thr Val Val Glu Thr Ser Ser Leu 370 375 380 LysCys Phe Ala Glu Thr Pro Asn Lys Lys Asn Lys Ile Thr Met Ile 385 390 395400 Ala Glu Pro Leu Glu Lys Gly Leu Ala Glu Asp Ile Glu Asn Glu Val 405410 415 Val Gln Ile Thr Trp Asn Arg Lys Lys Leu Gly Glu Phe Phe Gln Thr420 425 430 Lys Tyr Asp Trp Asp Leu Leu Ala Ala Arg Ser Ile Trp Ala PheGly 435 440 445 Pro Asp Ala Thr Gly Pro Asn Ile Leu Val Asp Asp Thr LeuPro Ser 450 455 460 Glu Val Asp Lys Ala Leu Leu Gly Ser Val Lys Asp SerIle Val Gln 465 470 475 480 Gly 4 33 PRT Homo Sapiens 4 Leu Gly Thr PheTrp Gly Asp Thr Leu Asn Cys Trp Met Leu Ser Ala 1 5 10 15 Phe Ser ArgTyr Ala Arg Cys Leu Ala Glu Gly His Asp Gly Pro Thr 20 25 30 Gln 5 99DNA Homo Sapiens 5 ttggggacat tttggggtga cacactgaac tgctggatgctatcagcatt tagtaggtat 60 gctcgatgtc ttgcagaagg acatgatggt cctacacag 99

What is claimed is:
 1. A purified polypeptide consisting of an aminoacid sequence as set forth in SEQ ID NO:3.
 2. A pharmaceuticalcomposition for promoting the survival and growth of motoneuronscomprising the polypeptide of claim 1 in a suitable carrier.
 3. Acomposition comprising the polypeptide of claim 1 and a suitablecarrier.
 4. A purified recombinant protein consisting of an amino acidsequence as set forth in SEQ ID NO:3, wherein the protein promotes thesurvival and growth of motoneurons.
 5. A purified fusion proteincomprising: a) an amino acid sequence as set forth in SEQ ID NO:3; andb) a heterologous amino acid sequence, which aids in the purification orisolation of the protein.